Combination antiretroviral therapy has resulted in a marked reduction in mortality and an increase in productive lifespan for HIV-infected individuals in developed nations. Despite great advances in HIV therapeutics, additional targets for antiretroviral therapy are needed. No current therapies disrupt the assembly phase of the retroviral life cycle. The overall goal of this research proposal is to identify novel antiretroviral compounds that inhibit HIV assembly. To do this, we develop novel assays that will allow identification of compounds that inhibit Gag-Gag interactions. The technical basis for the proposed assays is the use of fluorescence resonance energy transfer (FRET). We have proven that Gag-Gag FRET can be detected through the use of Gag-CFP and Gag-YFP fusion proteins. FRET occurs predominantly at the plasma membrane, as expected for the known assembly site of HIV particles. Using a cuvette-based fluorometer, Gag-Gag FRET is apparent as a demonstrable YFP emission curve following excitation at the CFP wavelength. We propose to use Gag-Gag FRET to develop two cell-based screening assays and an in vitro assay of Gag-Gag interaction. First, we will adapt the cuvette-based assay we have established to the detection of FRET in a microplate fluorometer. We propose a 96-well format for the development of screening assays. Stable cell line expressing Gag-CFP and Gag-YFP will be developed in order to standardize the assays. Second, we will develop a flow cytometry based assay for the detection of Gag-Gag interactions by FRET. A dual-laser FACSVantage will be equipped through the Vanderbilt Flow Cytometry Core Laboratory in conjunction with resources from this project to efficiently detect Gag-CFP/Gag-YFP FRET. The principal advantage of this technique is the ability to simultaneously monitor other cellular markers together with Gag-Gag interactions, and to detect quantitate events in distinct cellular populations. Finally, we will develop an in vitro assay for Gag-Gag interaction using the FRET readout. Recombinant proteins representing critical regions of Gag will be purified from bacteria. Labeling of recombinant Gag protein with an ideal fluorophore pair for FRET will be performed in vitro. Addition of Gag-donor fluorophore proteins to the wells containing Gag-acceptor fluorophore will initiate the kinetic assay for FRET. A fluorescence emission spectrum will be generated for each well over time, allowing selection of wells in which Gag-Gag interactions are inhibited. These assays will be used to screen libraries of compounds available from NIH, Vanderbilt, and industrial partners to identify candidate inhibitors of Gag-Gag interactions.